Modified binding proteins inhibiting the vegf-a receptor interaction

ABSTRACT

The present invention relates to binding proteins specific for VEGF-A, in particular to recombinant binding proteins comprising a polyethylene glycol moiety and a binding domain, which inhibits VEGF-Axxx binding to VEGFR-2. Examples of such recombinant binding proteins are proteins which comprise an ankyrin repeat domain with the desired binding specificity, and a polyethylene glycol moiety. The binding proteins are useful in the treatment of cancer and other pathological conditions, e.g. eye diseases such as age-related macular degeneration.

This is a divisional of application Ser. No. 15/811,841, filed Nov. 14,2017, now U.S. Pat. No. 10,646,542, which is a continuation ofapplication Ser. No. 15/015,902, filed Feb. 4, 2016, now U.S. Pat. No.9,849,158, which is a continuation of application Ser. No. 14/206,054,filed Mar. 12, 2014, now U.S. Pat. No. 9,289,466, which is a divisionalof application Ser. No. 13/643,618, filed Oct. 31, 2012, now U.S. Pat.No. 8,710,187, which is a 371 of PCT/EP2011/056824, filed Apr. 29, 2011,which claims priority to EP 10161685.2, filed Apr. 30, 2010, all ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to modified recombinant binding proteinsspecific for VEGF-A, as well as pharmaceutical compositions comprisingsuch proteins, and the use of such proteins in the treatment of tumorsand eye diseases.

BACKGROUND OF THE INVENTION

Angiogenesis, the growth of new blood vessels from pre-existingvasculature, is a key process in several pathological conditions,including tumor growth and eye diseases, in particular ocularneovascularization diseases such as age-related macular degeneration(AMD) or diabetic macular edema (DME) (Carmeliet, P., Nature 438,932-936, 2005). Vascular endothelial growth factors (VEGFs) stimulateangiogenesis and lymphangiogenesis by activating VEGF receptor (VEGFR)tyrosine kinases in endothelial cells (Ferrara, N., Gerber, H. P. andLeCouter, J., Nature Med. 9, 669-676, 2003).

The mammalian VEGF family consists of five glycoproteins referred to asVEGF-A, VEGF-B, VEGF-C, VEGF-D (also known as FIGF) and placenta growthfactor (PlGF, also known as PGF). VEGF-A has been shown to be aneffective target for anti-angiogenic therapy (Ellis, L. M. and Hicklin,D. J., Nature Rev. Cancer 8, 579-591, 2008). The VEGF-A ligands bind toand activate three structurally similar type III receptor tyrosinekinases, designated VEGFR-1 (also known as FLT1), VEGFR-2 (also known asKDR) and VEGFR-3 (also known as FLT4). The VEGF ligands have distinctivebinding specificities for each of these tyrosine kinase receptors, whichcontribute to their diversity of function.

In response to ligand binding, the VEGFR tyrosine kinases activate anetwork of distinct downstream signaling pathways. VEGFR-1 and VEGFR-2are primarily found on the vascular endothelium whereas VEGFR-3 ismostly found on the lymphatic endothelium. These receptors all have anextracellular domain, a single transmembrane region and a consensustyrosine kinase sequence interrupted by a kinase-insert domain. Morerecently neuropilin (NRP-1), originally identified as a receptor for thesemaphorin/collapsin family of neuronal guidance mediators, was shown toact as an isoform specific receptor for VEGF-A.

Various isoforms of VEGF-A are known that are generated by alternativesplicing from eight exons within the VEGF-A gene. All isoforms containexons 1-5 and the terminal exon, exon 8. Exons 6 and 7, which encodeheparin-binding domains, can be included or excluded. This gives rise toa family of proteins termed according to their amino acid number:VEGF-A165, VEGF-A121, VEGF-A189, and so on. Exon 8, however, containstwo 3′ splice sites in the nucleotide sequences, which can be used bythe cell to generate two families of isoforms with identical length, butdiffering C-terminal amino acid sequences (Varey, A. H. R. et al.,British J. Cancer 98, 1366-1379, 2008). VEGF-Axxx (“xxx” denotes theamino acid number of the mature protein), the pro-angiogenic family ofisoforms, is generated by use of the most proximal sequence in exon 8(resulting in the inclusion of exon 8a). The more recently describedanti-angiogenic VEGF-Axxxb isoforms are generated by the use of a distalsplice site, 66 bp further along the gene from the proximal splice site.This results in splicing out of exon 8a and the production of mRNAsequences that encode the VEGF-Axxxb family. VEGF-A165 is thepredominant pro-angiogenic isoform and is commonly overexpressed in avariety of human solid tumors. VEGF-A165b was the first of the exon8b-encoded isoforms identified and was shown to have anti-angiogeniceffects (Varey et al., loc. cit.; Konopatskaya, O. et al., MolecularVision 12, 626-632, 2006). It is an endogenous inhibitory form ofVEGF-A, which decreases VEGF-A induced proliferation and migration ofendothelial cells. Although it can bind to VEGFR-2, VEGF-A165b bindingdoes not result in receptor phosphorylation or activation of thedownstream signaling pathways.

There are several approaches to inhibiting VEGF-A signaling, includingneutralization of the ligand or receptor by antibodies, and blockingVEGF-A receptor activation and signaling with tyrosine kinaseinhibitors. VEGF-A targeted therapy has been shown to be efficacious asa single agent in AMD, DME, renal cell carcinoma and hepatocellularcarcinoma, whereas it is only of benefit when combined with chemotherapyfor patients with metastatic colorectal, non-small-cell lung andmetastatic breast cancer (Narayanan, R. et al., Nat Rev. Drug Discov. 5,815-816, 2005; Ellis and Hicklin, loc. cit).

Beside antibodies other binding domains can be used to neutralize aligand or a receptor (Skerra, A., J. Mol. Recog. 13, 167-187, 2000;Binz, H. K., Amstutz, P. and Plückthun, A., Nat. Biotechnol. 23,1257-1268, 2005). One such novel class of binding domains are based ondesigned repeat domains (WO 02/20565; Binz, H. K., Amstutz, P., Kohl,A., Stumpp, M. T., Briand, C., Forrer, P., Grütter, M. G., andPlückthun, A., Nat. Biotechnol. 22, 575-582, 2004). WO 02/20565describes how large libraries of repeat proteins can be constructed andtheir general application. Nevertheless, WO 02/20565 does neitherdisclose the selection of repeat domains with binding specificity forVEGF-Axxx nor concrete repeat sequence motifs of repeat domains thatspecifically bind to VEGF-Axxx.

Targeting VEGF-A with currently available therapeutics is not effectivein all patients, or for all diseases (e.g., EGFR-expressing cancers). Ithas even become increasingly apparent that the therapeutic benefitassociated with VEGF-A targeted therapy is complex and probably involvesmultiple mechanisms (Ellis and Hicklin, loc. cit.). For example,marketed anti-VEGF drugs, such as bevacizumab (AVASTIN®) or ranibizumab(LUCENTIS®) (see WO 96/030046, WO 98/045331 and WO 98/045332) or drugsin clinical development, such as VEGF-TRAP® (aflibercept) (WO 00/075319)do not distinguish between the pro- and anti-angiogenic forms of VEGF-A,so they do inhibit both. As a result, they inhibit angiogenesis, butalso deprive healthy tissues of an essential survival factor, namelyVEGF-Axxxb, resulting in cytotoxicity and dose-limiting side effects,which in turn limit efficacy. Side effects common to current anti-VEGF-Atherapies are gastrointestinal perforations, bleeding, hypertension,thromboembolic events and proteinuria (Kamba, T. and McDonald, D. M.,Br. J. Cancer 96, 1788-95, 2007). Another marketed anti-VEGF drug forthe treatment of AMD is pegaptanib (WO 98/018480; MACUGEN®, a registeredtrademark of Pfizer). Pegaptanib is a PEGylated anti-VEGF aptamer, asingle strand of nucleic acid that binds with specificity to the targetprotein. For the treatment of neovascular AMD there is ample evidencethat vision outcomes with LUCENTIS® (ranibizumab) are superior to thosewith MACUGEN® (pegaptanib), and there is no definitive evidence tosuggest a difference in safety between the drugs. As a result, MACUGEN®(pegaptanib) is not a commonly used therapy for this disease.

Overall, a need exists for improved anti-angiogenic agents for treatingcancer and other pathological conditions.

The technical problem underlying the present invention is to identifynovel anti-angiogenic agents, such as repeat domains with bindingspecificity to VEGF-Axxx, for an improved treatment of cancer and otherpathological conditions, e.g. eye diseases such as AMD or DME. Thesolution to this technical problem is achieved by providing theembodiments characterized in the claims.

SUMMARY OF THE INVENTION

The present invention relates to a recombinant binding proteincomprising an ankyrin repeat domain and a polyethylene glycol moiety ofat least 5 kDa molecular weight, wherein said ankyrin domain bindsVEGF-Axxx with a Kd below 10⁻⁹M and inhibits VEGF-Axxx binding toVEGFR-2.

In a preferred embodiment, the polyethylene glycol moiety is coupled toa single Cys residue of the binding domain.

The invention further relates to a pharmaceutical composition comprisingone or more of the above mentioned binding proteins or nucleic acidmolecules.

The invention further relates to a method of treatment of cancer andother pathological conditions, e.g. eye diseases such as AMD or DME,using the binding proteins of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Specific dog VEGF-A164 binding of selected designed ankyrinrepeat proteins. The interaction of selected clones with dog VEGF-A164(VEGF) and a negative control protein (MBP, Escherichia coli maltosebinding protein) is shown by crude extract ELISA. The biotinylated dogVEGF-A164 and MBP were immobilized over NEUTRAVIDIN®. The numbers referto single DARPin® clones selected in ribosome display against dogVEGF-A164 or the corresponding human VEGF-A165.

A=Absorbance. White bars indicate binding to dog VEGF-A164, black barsshow non-specific background binding to MBP.

FIGS. 2A and 2B. Spheroid outgrowth inhibition by a selected DARPin®protein. The length of sprouts in a spheroid outgrowth inhibition assayare shown in presence of various concentrations of (FIG. 2A) DARPin #30(amino acids 1 to 126 of SEQ ID NO:4), a DARPin® protein withspecificity to VEGF-Axxx, or (FIG. 2B) DARPin NC, a negative controlDARPin® protein with no specificity for VEGF-Axxx.

FIGS. 3A to 3D. Specific recognition of VEGF-A isoforms. Surface PlasmonResonance (SPR) analysis of binding proteins on VEGF-A isoforms. (FIG.3A) and (FIG. 3B): SPR analysis of AVASTIN® (bevacizumab). 250 nM ofAVASTIN® (bevacizumab) was applied to a flow cell with immobilized dogVEGF-A164 (FIG. 3A) or dog VEGF-A164b (FIG. 3B) for 100 seconds,followed by washing with buffer flow. (FIG. 3C) and (FIG. 3D): SPRanalysis of DARPin #27 (amino acids 1 to 159 of SEQ ID NO:1). 250 nM ofDARPin #27 was applied to a flow cell with immobilized dog VEGF-A164(FIG. 3C) or dog VEGF-A164b (FIG. 3D) for 100 seconds, followed bywashing with buffer flow.

RU=Resonance Units.

FIG. 4. Efficient inhibition of human VEGF-A165 in the rabbit eye.Vascular leakage rabbit model to show the efficacy of a DARPin® proteinin inhibiting human VEGF-A165 in the eye in comparison to LUCENTIS®(ranibizumab). At day 1 either PBS, DARPin #30 or LUCENTIS®(ranibizumab) is applied by an intravitreal injection into one eye ofeach rabbit (treated eye). At day 4 or day 30 both eyes of each rabbitwere challenged by intravitreal injection of 500 ng of human VEGF-A165.All eyes were evaluated 48 hours after the VEGF-A165 injection bymeasuring the fluorescein content in the vitreous and retina of all eyesone hour after intravenous injection of sodium fluorescein.

R=ratio of fluorescein measurements treated eye/untreated eye. Standarddeviations are shown by an error bar. 4-PBS=ratio 4 days after injectionof PBS (control); 4-D=ratio 4 days after injection of DARPin #30;30-D=ratio 30 days after injection of DARPin #30; 4-L=ratio 4 days afterinjection of LUCENTIS® (ranibizumab); 30-L=ratio 30 days after injectionof LUCENTIS® (ranibizumab).

DETAILED DESCRIPTION OF THE INVENTION

Mammalian VEGF-A exists as two families of alternative spliced isoforms:(i) the pro-angiogenic “VEGF-Axxx” isoforms generated by proximalsplicing of exon 8 and (ii) the anti-angiogenic “VEGF-Axxxb” isoformsgenerated by distal splicing of exon 8. Preferably, the binding domainaccording to the invention is specific for the pro-angiogenic VEGF-Axxxof dog, rabbit, monkey or human origin. More preferably, the bindingdomain according to the invention is specific for the pro-angiogenicVEGF-Axxx of human origin. Most preferred, the binding domain accordingto the invention is specific for human VEGF-A165.

The term “protein” refers to a polypeptide, wherein at least part of thepolypeptide has, or is able to acquire a defined three-dimensionalarrangement by forming secondary, tertiary, or quaternary structureswithin and/or between its polypeptide chain(s). If a protein comprisestwo or more polypeptides, the individual polypeptide chains may belinked non-covalently or covalently, e.g. by a disulfide bond betweentwo polypeptides. A part of a protein, which individually has, or isable to acquire a defined three-dimensional arrangement by formingsecondary or tertiary structures, is termed “protein domain”. Suchprotein domains are well known to the practitioner skilled in the art.

The term “recombinant” as used in recombinant protein, recombinantprotein domain and the like, means that said polypeptides are producedby the use of recombinant DNA technologies well known by thepractitioner skilled in the relevant art. For example, a recombinant DNAmolecule (e.g. produced by gene synthesis) encoding a polypeptide can becloned into a bacterial expression plasmid (e.g. pQE30, Qiagen). Whensuch a constructed recombinant expression plasmid is inserted into abacteria (e.g. E. coli), this bacteria can produce the polypeptideencoded by this recombinant DNA. The correspondingly producedpolypeptide is called a recombinant polypeptide.

The term “polypeptide tag” refers to an amino acid sequence attached toa polypeptide/protein, wherein said amino acid sequence is useful forthe purification, detection, or targeting of said polypeptide/protein,or wherein said amino acid sequence improves the physicochemicalbehavior of the polypeptide/protein, or wherein said amino acid sequencepossesses an effector function. The individual polypeptide tags,moieties and/or domains of a binding protein may be connected to eachother directly or via polypeptide linkers. These polypeptide tags areall well known in the art and are fully available to the person skilledin the art. Examples of polypeptide tags are small polypeptidesequences, for example, His, myc, FLAG, or Strep-tags or moieties suchas enzymes (for example enzymes like alkaline phosphatase), which allowthe detection of said polypeptide/protein, or moieties which can be usedfor targeting (such as immunoglobulins or fragments thereof) and/or aseffector molecules.

The term “polypeptide linker” refers to an amino acid sequence, which isable to link, for example, two protein domains, a polypeptide tag and aprotein domain, a protein domain and a non-polypeptide moiety such aspolyethylene glycol or two sequence tags. Such additional domains, tags,non-polypeptide moieties and linkers are known to the person skilled inthe relevant art. A list of example is provided in the description ofthe patent application WO 02/20565. Particular examples of such linkersare glycine-serine-linkers and proline-threonine-linkers of variablelengths; preferably, said linkers have a length between 2 and 24 aminoacids; more preferably, said linkers have a length between 2 and 16amino acids.

In the context of the present invention, the term “polypeptide” relatesto a molecule consisting of one or more chains of multiple, i.e. two ormore, amino acids linked via peptide bonds. Preferably, a polypeptideconsists of more than eight amino acids linked via peptide bonds.

The term “polymer moiety” refers to either a proteinaceous polymermoiety or a non-proteinaceous polymer moiety. A “proteinaceous polymermoiety” preferably is a polypeptide that does not form a stable tertiarystructure while not forming more than 10% (preferably, not more than 5%;also preferred, not more than 2%; even more preferably, not more than1%; and most preferably, no detectable amounts, as determined by sizeexclusion chromatography (SEC)) of oligomers or aggregates when storedat a concentration of about 0.1 mM in PBS at RT for one month. Suchproteinaceous polymer moieties run at an apparent molecular weight inSEC that is higher than their effective molecular weight when usingglobular proteins as molecular weight standards for the SEC. Preferably,the apparent molecular weight of said proteinaceous polymer moietiesdetermined by SEC is 1.5×, 2× or 2.5× higher than their effectivemolecular weight calculated from their amino acid sequence. Alsopreferably, the apparent molecular weights of said non-proteinaceouspolymer moieties determined by SEC is 2×, 4× or 8× higher than theireffective molecular weight calculated from their molecular composition.Preferably, more than 50%, 70% or even 90% of the amino acids of saidproteinaceous polymer moiety do not form stable secondary structures ata concentration of about 0.1 mM in PBS at RT as determined by CircularDichroism (CD) measurements. Most preferably, said proteinaceous polymershows a typical near UV CD-spectra of a random coil conformation. SuchCD analyses are well known to the person skilled in the art. Alsopreferable are proteinaceous polymer moieties that consist of more than50, 100, 200, 300, 400, 500, 600, 700 or 800 amino acids. Examples ofproteinaceous polymer moieties are XTEN® (a registered trademark ofAmunix; WO 07/103515) polypeptides, or polypeptides comprising proline,alanine and serine residues as described in WO 08/155134. Suchproteinaceous polymer moieties can be covalently attached to, forexample, a binding domain of the invention by the generation of geneticfusion polypeptides using standard DNA cloning technologies, followed bytheir standard expression and purification. Examples of binding proteinscomprising a repeat domain binding VEGF-Axxx and such a proteinaceouspolymer moiety are shown in SEQ ID NO:1 and SEQ ID NO:4. The amino acidpositions from 1 to 159 of SEQ ID NO:1 correspond to the repeat domainand the amino acid position 161 to 1′025 of SEQ ID NO:1 correspond tothe proteinaceous polymer moiety. The amino acid positions from 1 to 126of SEQ ID NO:4 correspond to the repeat domain and the amino acidpositions 131 to 640 of SEQ ID NO:4 correspond to the proteinaceouspolymer moiety.

A polymer moiety of the invention may vary widely in molecular weight(i.e. from about 1 kDa to about 150 kDa). Preferably, the polymer moietyhas a molecular weight of at least 2, 5, 10, 20, 30, 50, 70 or 100 kDa.

Preferably, said polymer moiety is connected by a polypeptide linker toa binding domain. Examples of such polypeptide linkers are the aminoacids 1 to 8 of SEQ ID NO:8 and SEQ ID NO:9.

Examples of non-proteinaceous polymer moieties are hydroxyethyl starch(HES), polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylene. The term “PEGylated” means that a PEG moiety iscovalently attached to, for example, a polypeptide of the invention.Examples of repeat proteins containing a polypeptide linker between therepeat domain and a C-terminal Cys residue useful for binding anon-proteinaceous polymer moiety are SEQ ID NO:2, 3, 5, 6 and 7.

In a specific embodiment, a PEG moiety or any other non-proteinaceouspolymer can, e.g., be coupled to a cysteine thiol via a maleimide linkerwith the cysteine being coupled via a peptide linker to the N- orC-terminus of a binding domain as described herein (e.g. SEQ ID NO:3).

The term “binding protein” refers to a protein comprising one or morebinding domains and one or more polymer moieties as further explainedbelow. Preferably, said binding protein comprises up to four bindingdomains. More preferably, said binding protein comprises up to twobinding domains. Most preferably, said binding protein comprises onlyone binding domain. Furthermore, any such binding protein may compriseadditional protein domains that are not binding domains, multimerizationmoieties, polypeptide tags, polypeptide linkers and/or a single Cysresidue. Examples of multimerization moieties are immunoglobulin heavychain constant regions which pair to provide functional immunoglobulinFc domains, and leucine zippers or polypeptides comprising a free thiolwhich forms an intermolecular disulfide bond between two suchpolypeptides. The single Cys residue may be used for conjugating othermoieties to the polypeptide, for example, by using the maleimidechemistry well known to the person skilled in the art.

Preferably, said binding protein comprises up to four polymer moieties.More preferably, said binding protein comprises up to two polymermoieties. Most preferably, said binding protein comprises only onepolymer moiety.

Also preferably, said binding protein has an apparent molecular weightof at least 70, 100, 200, 300, 500 or 800 kDa when analyzed at aconcentration of 0.1 mM in PBS at RT by SEC using globular proteins asmolecular weight standards.

The term “binding domain” means a protein domain exhibiting the same“fold” (three-dimensional arrangement) as a protein scaffold and havinga predetermined property, as defined below. Such a binding domain may beobtained by rational, or most commonly, combinatorial proteinengineering techniques, skills which are known in the art (Skerra, 2000,loc. cit.; Binz et al., 2005, loc. cit.). For example, a binding domainhaving a predetermined property can be obtained by a method comprisingthe steps of (a) providing a diverse collection of protein domainsexhibiting the same fold as a protein scaffold as defined further below;and (b) screening said diverse collection and/or selecting from saiddiverse collection to obtain at least one protein domain having saidpredetermined property. The diverse collection of protein domains may beprovided by several methods in accordance with the screening and/orselection system being used, and may comprise the use of methods wellknown to the person skilled in the art, such as phage display orribosome display.

The term “protein scaffold” means a protein with exposed surface areasin which amino acid insertions, substitutions or deletions are highlytolerable. Examples of protein scaffolds that can be used to generatebinding domains of the present invention are antibodies or fragmentsthereof such as single-chain Fv or Fab fragments, protein A fromStaphylococcus aureus, the bilin binding protein from Pieris brassicaeor other lipocalins, ankyrin repeat proteins or other repeat proteins,and human fibronectin. Protein scaffolds are known to the person skilledin the art (Binz et al., 2005, loc. cit.; Binz et al., 2004, loc. cit.).

The term “predetermined property” refers to a property such as bindingto a target, blocking of a target, activation of a target-mediatedreaction, enzymatic activity, and related further properties. Dependingon the type of desired property, one of ordinary skill will be able toidentify format and necessary steps for performing screening and/orselection of a binding domain with the desired property. Preferably,said predetermined property is binding to a target.

Preferably, the binding protein of the invention is not an antibody or afragment thereof, such as Fab or scFv fragments. Antibodies andfragments thereof are well known to the person skilled in the art.

Also preferably, the binding domain of the invention does not comprisean immunoglobulin fold as present in antibodies and/or the fibronectintype III domain. An immunoglobulin fold is a common all-β protein foldthat consists of a 2-layer sandwich of about 7 anti-parallel β-strandsarranged in two β-sheets. Immunoglobulin folds are well known to theperson skilled in the art. For example, such binding domains comprisingan immunoglobulin fold are described in WO 07/080392 or WO 08/097497.

Further preferably, the binding domain of the invention does notcomprise an immunoglobulin-like domain as found in VEGFR-1 or VEGFR-2.Such binding domains are described in WO 00/075319.

A preferred binding domain is a binding domain having anti-angiogeniceffects. The anti-angiogenic effect of a binding domain can bedetermined by assays well know to the person skilled in the art, such asthe sprouting assay of HUVEC spheroids described in Example 2.

Further preferred is a binding domain comprising between 70 and 300amino acids, in particular between 100 and 200 amino acids.

Further preferred is a binding domain devoid of a free Cys residue. Afree Cys residue is not involved in the formation of a disulfide bond.Even more preferred is a binding domain free of any Cys residue.

A preferred binding domain of the invention is a repeat domain or adesigned repeat domain, preferably as described in WO 02/20565.

A particularly preferred binding domain is a designed ankyrin repeatdomain (Binz, H. K. et al., 2004, loc. cit.), preferably as described inWO 02/20565. Examples of designed ankyrin repeat domains are shown inthe Examples.

The definitions hereinafter for repeat proteins are based on those inpatent application WO 02/20565. Patent application WO 02/20565 furthercontains a general description of repeat protein features, techniquesand applications.

The term “repeat proteins” refers to a protein comprising one or morerepeat domains. Preferably, each of said repeat proteins comprises up tofour repeat domains. More preferably, each of said repeat proteinscomprises up to two repeat domains. Most preferably, each of the repeatproteins comprises only one repeat domain. Furthermore, said repeatprotein may comprise additional non-repeat protein domains, polypeptidetags and/or polypeptide linkers.

The term “repeat domain” refers to a protein domain comprising two ormore consecutive repeat units (modules) as structural units, whereinsaid structural units have the same fold, and stack tightly to create,for example, a superhelical structure having a joint hydrophobic core.

The term “designed repeat protein” and “designed repeat domain” refer toa repeat protein or repeat domain, respectively, obtained as the resultof the inventive procedure explained in patent application WO 02/20565.Designed repeat proteins and designed repeat domains are synthetic andnot from nature. They are man-made proteins or domains, respectively,obtained by expression of correspondingly designed nucleic acids.Preferably, the expression is done in eukaryotic or prokaryotic cells,such as bacterial cells, or by using a cell-free in vitro expressionsystem.

The term “structural unit” refers to a locally ordered part of apolypeptide, formed by three-dimensional interactions between two ormore segments of secondary structure that are near one another along thepolypeptide chain. Such a structural unit exhibits a structural motif.The term “structural motif” refers to a three-dimensional arrangement ofsecondary structure elements present in at least one structural unit.Structural motifs are well known to the person skilled in the art.Structural units alone are not able to acquire a definedthree-dimensional arrangement; however, their consecutive arrangement,for example as repeat modules in a repeat domain, leads to a mutualstabilization of neighboring units resulting in a superhelicalstructure.

The term “repeat unit” refers to amino acid sequences comprising repeatsequence motifs of one or more naturally occurring repeat proteins,wherein said “repeat units” are found in multiple copies, and whichexhibit a defined folding topology common to all said motifs determiningthe fold of the protein. Examples of such repeat units are armadillorepeat units, leucine-rich repeat units, ankyrin repeat units,tetratricopeptide repeat units, HEAT repeat units, and leucine-richvariant repeat units. Naturally occurring proteins containing two ormore such repeat units are referred to as “naturally occurring repeatproteins”. The amino acid sequences of the individual repeat units of arepeat protein may have a significant number of mutations,substitutions, additions and/or deletions when compared to each other,while still substantially retaining the general pattern, or motif, ofthe repeat units.

Preferably, the repeat units used for the deduction of a repeat sequencemotif are homologous repeat units obtained from repeat domains selectedon a target, for example as described in Example 1 and having the sametarget-specificity.

The term “repeat sequence motif” refers to an amino acid sequence, whichis deduced from one or more repeat units. Preferably, said repeat unitsare from repeat domains having binding specificity for the same target.

The term “folding topology” refers to the tertiary structure of saidrepeat units. The folding topology will be determined by stretches ofamino acids forming at least parts of α-helices or β-sheets, or aminoacid stretches forming linear polypeptides or loops, or any combinationof α-helices, β-sheets and/or linear polypeptides/loops.

The term “consecutive” refers to an arrangement, wherein the repeatunits or repeat modules are arranged in tandem. In designed repeatproteins, there are at least 2, usually about 2 to 6, in particular atleast about 6, frequently 20 or more repeat units. In most cases, repeatunits will exhibit a high degree of sequence identity (same amino acidresidues at corresponding positions) or sequence similarity (amino acidresidues being different, but having similar physicochemicalproperties), and some of the amino acid residues might be key residuesbeing strongly conserved in the different repeat units found innaturally occurring proteins. However, a high degree of sequencevariability by amino acid insertions and/or deletions, and/orsubstitutions between the different repeat units found in naturallyoccurring proteins will be possible as long as the common foldingtopology is maintained.

Methods for directly determining the folding topology of repeat proteinsby physicochemical means such as X-ray crystallography, NMR or CDspectroscopy, are well known to the practitioner skilled in the art.Methods for identifying and determining repeat units or repeat sequencemotifs or for identifying families of related proteins comprising suchrepeat units or motifs, such as homology searches (BLAST® etc.), arewell established in the field of bioinformatics, and are well known tothe practitioner in the art. The step of refining an initial repeatsequence motif may comprise an iterative process.

The term “repeat modules” refers to the repeated amino acid sequences ofthe designed repeat domains, which are originally derived from therepeat units of naturally occurring repeat proteins. Each repeat modulecomprised in a repeat domain is derived from one or more repeat units ofthe family or subfamily of naturally occurring repeat proteins, e.g. thefamily of armadillo repeat proteins or ankyrin repeat proteins.

“Repeat modules” may comprise positions with amino acid residues presentin all copies of corresponding repeat modules (“fixed positions”) andpositions with differing or “randomized” amino acid residues(“randomized positions”).

The term “capping module” refers to a polypeptide fused to the N- orC-terminal repeat module of a repeat domain, wherein said capping moduleforms tight tertiary interactions with said repeat module therebyproviding a cap that shields the hydrophobic core of said repeat moduleat the side not in contact with the consecutive repeat module from thesolvent. Said N- and/or C-terminal capping module may be, or may bederived from, a capping unit or other domain found in a naturallyoccurring repeat protein adjacent to a repeat unit. The term “cappingunit” refers to a naturally occurring folded polypeptide, wherein saidpolypeptide defines a particular structural unit which is N- orC-terminally fused to a repeat unit, wherein said polypeptide formstight tertiary interactions with said repeat unit thereby providing acap that shields the hydrophobic core of said repeat unit at one sidefrom the solvent. Such capping units may have sequence similarities tosaid repeat sequence motif. Capping modules and capping repeats aredescribed in WO 02/020565. For example, the N-terminal capping module ofSEQ ID NO:2 is encoded by the amino acids from position 1 to 32. Alsopreferred is such an N-terminal capping module having a glycine oraspartate residue at position 5.

The term “target” refers to an individual molecule such as a nucleicacid molecule, a polypeptide or protein, a carbohydrate, or any othernaturally occurring molecule, including any part of such individualmolecule, or complexes of two or more of such molecules. The target maybe a whole cell or a tissue sample, or it may be any non-naturalmolecule or moiety. Preferably, the target is a naturally occurring ornon-natural polypeptide or a polypeptide containing chemicalmodifications, for example modified by natural or non-naturalphosphorylation, acetylation, or methylation. In the particularapplication of the present invention, the target is VEGF-Axxx orVEGFR-2.

The term “consensus sequence” refers to an amino acid sequence, whereinsaid consensus sequence is obtained by structural and/or sequencealigning of multiple repeat units. Using two or more structural and/orsequence aligned repeat units, and allowing for gaps in the alignment,it is possible to determine the most frequent amino acid residue at eachposition. The consensus sequence is that sequence which comprises theamino acids which are most frequently represented at each position. Inthe event that two or more amino acids are represented above-average ata single position, the consensus sequence may include a subset of thoseamino acids. Said two or more repeat units may be taken from the repeatunits comprised in a single repeat protein, or from two or moredifferent repeat proteins.

Consensus sequences and methods to determine them are well known to theperson skilled in the art.

A “consensus amino acid residue” is the amino acid found at a certainposition in a consensus sequence. If two or more, e.g. three, four orfive, amino acid residues are found with a similar probability in saidtwo or more repeat units, the consensus amino acid may be one of themost frequently found amino acids or a combination of said two or moreamino acid residues.

Further preferred are non-naturally occurring binding proteins orbinding domains.

The term “non-naturally occurring” means synthetic or not from nature,more specifically, the term means made from the hand of man. The term“non-naturally occurring binding protein” or “non-naturally occurringbinding domain” means that said binding protein or said binding domainis synthetic (i.e. produced by chemical synthesis from amino acids) orrecombinant and not from nature. “Non-naturally occurring bindingprotein” or “non-naturally occurring binding domain” is a man-madeprotein or domain, respectively, obtained by expression ofcorrespondingly designed nucleic acids. Preferably, the expression isdone in eukaryotic or bacterial cells, or by using a cell-free in vitroexpression system. Further, the term means that the sequence of saidbinding protein or said binding domain is not present as anon-artificial sequence entry in a sequence database, for example inGenBank, EMBL-Bank or Swiss-Prot. These databases and other similarsequence databases are well known to the person skilled in the art.

A binding domain can inhibit VEGF-Axxx binding to VEGFR-2 either bybinding to VEGF-Axxx or by binding to VEGFR-2 in a way that the apparentdissociation constant (K_(d)) between VEGF-Axxx and VEGFR-2 is increasedmore than 10²-fold, preferably more than 10³-fold, more preferably morethan 10⁴-fold, more preferably more than 10⁵-fold, and most preferablymore than 10⁶-fold. Preferably, the K_(d) for the interaction of thebinding domain to either VEGF-Axxx or VEGFR-2 is below 10⁻⁷M, preferablybelow 10⁻⁸M, more preferably below 10⁻⁹M, more preferably below 10⁻¹⁰M,and most preferably below 10⁻¹¹M. Methods, to determine dissociationconstants of protein-protein interactions, such as surface plasmonresonance (SPR) based technologies, are well known to the person skilledin the art.

A preferred binding domain binds VEGF-Axxx. Even more preferred is abinding domain that binds human VEGF-A165.

The term “PBS” means a phosphate buffered water solution containing 137mM NaCl, 10 mM phosphate and 2.7 mM KCl and having a pH of 7.4.

Preferred is a binding protein and/or binding domain that does not loseits native three-dimensional structure upon incubation in PBS containing100 mM dithiothreitol (DTT) for 1 or 10 hours at 37° C.

In one particular embodiment the invention relates to a binding proteincomprising a binding domain inhibiting VEGF-Axxx binding to VEGFR-2 andhaving the indicated or preferred midpoint denaturation temperature andnon-aggregating properties as defined above, wherein said bindingprotein inhibits sprouting of HUVEC spheroids with an IC₅₀ value below100 nM.

The term “HUVEC” means human umbilical vein endothelial cells, which canbe isolated from normal human umbilical vein and which are responsive toVEGF-A stimulation. Assays to measure the sprouting of HUVEC spheroids,such as that described in Example 2, are well known to the personskilled in the art.

An IC₅₀ value is the concentration of a substance, such as a bindingprotein or binding domain, which is required for 50% inhibition in vitroof an experimental determined parameter, such as the sprouting of HUVECspheroids. IC₅₀ values can be readily determined by the person skilledin the art (Korff T. and Augustin H. G., J. Cell Biol. 143(5), 1341-52,1998).

Preferred is a binding protein and/or binding domain that inhibits thesprouting of HUVEC spheroid with an IC₅₀ value below 10 nM, preferablybelow 1 nM, more preferably below 0.1 nM, and most preferably below 0.05nM.

Further preferred is a monomeric binding protein and/or binding domainthat inhibits the sprouting of HUVEC spheroids with an IC₅₀ value lowerthan the corresponding IC₅₀ value of ranibizumab (LUCENTIS®, aregistered trademark of Genentech), bevacizumab (AVASTIN®, a registeredtrademark of Genentech), aflibercept (VEGF-TRAP®, a registered trademarkof Regeneron), or pegaptanib (MACUGEN®, a registered trademark ofPfizer).

The K_(d) for the interaction of a preferred binding domain to VEGF-B,VEGF-C, VEGF-D, PlGF or PDGF is above 1 nM, preferably above 10 nM, morepreferably above 10² nM, even more preferably above 10³ nM, and mostpreferably above 10⁴ nM.

Preferably, VEGF-Axxx is either dog VEGF-A164 or simian VEGF-A165 orhuman VEGF-A165, and VEGF-Axxxb is either dog VEGF-A164b or simianVEGF-A165b or human VEGF-A165b.

Another preferred embodiment is a recombinant binding protein comprisinga binding domain, wherein said binding domain inhibits VEGF-Axxx bindingto VEGFR-2 and wherein said binding domain is a repeat domain or adesigned repeat domain. Such a repeat domain may comprise one, two,three or more internal repeat modules that will participate in bindingto VEGF-Axxx. Preferably, such a repeat domain comprises an N-terminalcapping module, two to four internal repeat modules, and a C-terminalcapping module. Preferably, said binding domain is an ankyrin repeatdomain or designed ankyrin repeat domain.

A preferred recombinant binding protein comprises a binding domain asdescribed herein, conjugated to a polyethylene glycol (PEG) moiety,preferably wherein said PEG moiety is coupled to a single Cys residue ofsaid binding domain. Preferably, said Cys residue is geneticallyintroduced at the C-terminal end of said binding domain. The PEG moietycan then be coupled by chemical means, for example, by using maleimidechemistry well known to the person skilled in the art. Examples of suchbinding proteins comprising a PEG moiety conjugated to a single Cysresidue are given in the Examples.

A preferred embodiment of the invention comprises a recombinant bindingprotein comprising a binding domain as described herein, wherein saidbinding domain is conjugated at its C-terminus via a peptide bond to SEQID NO:8, which is in turn conjugated at the C-terminal cysteine thiol toa maleimide-coupled PEG, such asα-[3-(3-maleimido-1-oxopropyl)amino]propyl-ω-methoxy-polyoxyethylene(NOF, SUNBRIGHT® ME-200MA (20 kD) or SUNBRIGHT® ME-400MA (40 kD)). Inone embodiment theα-[3-(3-maleimido-1-oxopropyl)amino]propyl-ω-methoxy-polyoxyethylene hasa molecular weight of at least about 2, 5, 10, 20, 30, 40, 50, 70, or100 kD. In certain embodiments theα-[3-(3-maleimido-1-oxopropyl)amino]propyl-ω-methoxy-polyoxyethylene hasa molecular weight of at least about 20 or at least about 40 kD.

Another preferred embodiment is a recombinant binding protein as definedabove comprising at least one repeat domain with binding specificity forVEGF-Axxx, wherein said repeat domain competes for binding to VEGF-Axxxwith a repeat domain selected from the group of the repeat domains ofSEQ ID NO:1 to 7. Preferably, said repeat domain competes for binding toVEGF-Axxx with the repeat domain of SEQ ID NO:1 or 3. More preferably,said repeat domain competes for binding to VEGF-Axxx with the repeatdomain of SEQ ID NO:3.

The term “compete for binding” means the inability of two differentbinding domains of the invention to bind simultaneously to the sametarget, while both are able to bind the same target individually. Thus,such two binding domains compete for binding to said target. Methods,such as competition ELISA or competition SPR measurements (e.g. by usingthe Proteon instrument from BioRad), to determine if two binding domainscompete for binding to a target are well known to the practitioner inthe art.

A recombinant binding protein that competes for binding to VEGF-Axxxwith a selected repeat protein can be identified by methods well knownto the person skilled in the art, such as a competition Enzyme-LinkedImmunoSorbent Assay (ELISA).

Another preferred embodiment is a recombinant binding protein comprisinga repeat domain with binding specificity for VEGF-Axxx selected from thegroup consisting of the repeat domains of SEQ ID NO:1 to 7. Preferably,said repeat domain is selected from the repeat domains of SEQ ID NO:2 or3. More preferably, said repeat domain is the repeat domain of SEQ IDNO:3.

One ore more polyethylene glycol moieties may be attached at differentpositions in the binding protein, and such attachment may be achieved byreaction with amines, thiols or other suitable reactive groups.Attachment of polyethylene glycol moieties (PEGylation) may besite-directed, wherein a suitable reactive group is introduced into theprotein to create a site where PEGylation preferentially occurs, or isoriginally present in the binding protein. The thiol group may bepresent in a cysteine residue; and the amine group may be, for example,a primary amine found at the N-terminus of the polypeptide or an aminegroup present in the side chain of an amino acid, such as lysine orarginine. In a preferred embodiment, the binding protein is modified soas to have a cysteine residue at a desired position, permitting sitedirected PEGylation on the cysteine, for example by reaction with apolyethylene glycol derivative carrying a maleimide function. Thepolyethylene glycol moiety may vary widely in molecular weight (i.e.from about 1 kDa to about 100 kDa) and may be branched or linear.Preferably, the polyethylene glycol has a molecular weight of about 1 toabout 50 kDa, preferably about 10 to about 40 kDa, even more preferablyabout 15 to about 30 kDa, and most preferably about 20 kDa.

In a further embodiment, the invention relates to nucleic acid moleculesencoding the particular recombinant binding proteins. Further, a vectorcomprising said nucleic acid molecule is considered.

Further, a pharmaceutical composition comprising one or more of theabove mentioned binding proteins, in particular recombinant bindingproteins comprising repeat domains, or nucleic acid molecules encodingthe particular recombinant binding proteins, and optionally apharmaceutical acceptable carrier and/or diluent is considered.

Pharmaceutical acceptable carriers and/or diluents are known to theperson skilled in the art and are explained in more detail below. Evenfurther, a diagnostic composition comprising one or more of the abovementioned recombinant binding proteins, in particular binding proteinscomprising repeat domains, is considered.

The binding protein of the invention suppresses or prevents VEGF inducedpathological angiogenesis, vascular leakage (edema), pulmonaryhypertension, tumor formation and/or inflammatory disorders. With“suppression” it is understood that the recombinant protein prevents thementioned pathologies to some extent, e.g. to 10% or 20%, morepreferably 50%, in particular 70%, 80% or 90%, or even 95%.

The term “edema” means a condition that is caused by vascular leakage.Vasodilation and increased permeability during inflammation can bepredominant pathogenetic mechanisms. For instance, edema contributes toinfarct expansion after stroke and may cause life-threateningintracranial hypertension in cancer patients. Further, extravasation ofplasma proteins favors metastatic spread of occult tumors, and airwaycongestion may cause fatal asthmatic attacks. The increased vascularleakage which occurs during inflammation can lead to respiratorydistress, ascites, peritoneal sclerosis (in dialysis patients), adhesionformation (abdominal surgery) and metastatic spreading.

The term “angiogenesis” means a fundamental process by which new bloodvessels are formed. The primary angiogenic period in humans takes placeduring the first three months of embryonic development but angiogenesisalso occurs as a normal physiological process during periods of tissuegrowth, such as an increase in muscle or fat and during the menstrualcycle and pregnancy.

The term “pathological angiogenesis” refers to the formation and growthof blood vessels during the maintenance and the progression of severaldisease states. Particular examples of pathological angiogenesis arefound in blood vessels (atherosclerosis, hemangioma,hemangioendothelioma), bone and joints (rheumatoid arthritis, synovitis,bone and cartilage destruction, osteomyelitis, pannus growth, osteophyteformation, neoplasms and metastasis), skin (warts, pyogenic granulomas,hair growth, Kaposi's sarcoma, scar keloids, allergic edema, neoplasms),liver, kidney, lung, ear and other epithelia (inflammatory andinfectious processes including hepatitis, glomerulonephritis, pneumonia;and asthma, nasal polyps, otitis, transplantation disorders, liverregeneration disorders, neoplasms and metastasis), uterus, ovary andplacenta (dysfunctional uterine bleeding due to intra-uterinecontraceptive devices, follicular cyst formation, ovarianhyperstimulation syndrome, endometriosis, neoplasms), brain, nerves andeye (retinopathy of prematurity, diabetic retinopathy, choroidal andother intraocular disorders, leukomalacia, neoplasms and metastasis),heart and skeletal muscle due to work overload, adipose tissue(obesity), endocrine organs (thyroiditis, thyroid enlargement, pancreastransplantation disorders), hematopoiesis (Kaposi syndrome in AIDS),hematologic malignancies (leukemias), and lymph vessels (tumormetastasis, lymphoproliferative disorders).

The term “retinal ischemic diseases” means that the retina's supply ofblood and oxygen is decreased, the peripheral portions of the retinalose their source of nutrition and stop functioning properly. Aparticular example of a retinal ischemic disease is retinopathy. Commondiseases which lead to retinopathy are diabetic retinopathy, centralretinal vein occlusion, stenosis of the carotid artery, and sickle cellretinopathy. Diabetic retinopathy is a major cause of visual loss indiabetic patients. In the ischemic retina the growth of new bloodvessels occurs (neovascularisation). These vessels often grow on thesurface of the retina, at the optic nerve, or in the front of the eye onthe iris. The new vessels cannot replace the flow of necessary nutrientsand, instead, can cause many problems such as vitreous hemorrhage,retinal detachment, and uncontrolled glaucoma. These problems occurbecause new vessels are fragile and are prone to bleed. If caught in itsearly stages, proliferative diabetic retinopathy can sometimes bearrested with panretinal photocoagulation. However, in some cases,vitrectomy surgery is the only option.

Beside these retinopathies, vascular diseases of the eye also includeocular neovascularization diseases, such as macular degeneration anddiabetic macular edema (DME). Macular degeneration results from theneovascular growth of the choroid vessel underneath the macula. Thereare two types of macular degeneration: dry and wet. While wet maculardegeneration only comprises 15% of all macular degeneration, nearly allwet macular degeneration leads to blindness. In addition, wet maculardegeneration nearly always results from dry macular degeneration. Onceone eye is affected by wet macular degeneration, the condition almostalways affects the other eye. Wet macular degeneration is often calledage-related wet macular degeneration of wet-AMD as it is mostly found inelderly persons.

Diabetic retinopathy (DR) and DME are leading causes of blindness in theworking-age population of most developed countries. The increasingnumber of individuals with diabetes worldwide suggests that DR and DMEwill continue to be major contributors to vision loss and associatedfunctional impairment for years to come. Several biochemical mechanisms,including protein kinase C-β activation, increased vascular endothelialgrowth factor production, oxidative stress, and accumulation ofintracellular sorbitol and advanced glycosylation end products, maycontribute to the vascular disruptions that characterize DR/DME. Theinhibition of these pathways holds the promise of intervention for DRand DME.

The term “pulmonary hypertension” means a disorder in which the bloodpressure in the pulmonary arteries is abnormally high. In the absence ofother diseases of the heart or lungs it is called primary pulmonaryhypertension. Diffuse narrowing of the pulmonary arterioles occurs as aresult of pathological arteriogenesis followed by pulmonary hypertensionas a response to the increased resistance to blood flow. The incidenceis 8 out of 100′000 people. However, pulmonary hypertension can alsooccur as a complication of Chronic Obstructive Pulmonary Diseases (COPD)such as emphysema, chronic bronchitis or diffuse interstitial fibrosisand in patients with asthmatiform COPD. The incidence of COPD isapproximately 5 out of 10′000 people.

Furthermore the binding proteins of the invention can be used to treatinflammation and more specifically inflammatory disorders.

The term “inflammation” as used herein means, the local reaction toinjury of living tissues, especially the local reaction of the smallblood vessels, their contents, and their associated structures. Thepassage of blood constituents through the vessel walls into the tissuesis the hallmark of inflammation, and the tissue collection so formed istermed the exudates or edema. Any noxious process that damages livingtissue, e.g. infection with bacteria, excessive heat, cold, mechanicalinjury such as crushing, acids, alkalis, irradiation, or infection withviruses can cause inflammation irrespective of the organ or tissueinvolved. It should be clear that diseases classified as “inflammatorydiseases” and tissue reactions ranging from burns to pneumonia, leprosy,tuberculosis, and rheumatoid arthritis are all “inflammations”.

The binding proteins according to the invention can be used to treattumor formation. The term “tumor” means a mass of abnormal tissue thatarises without obvious cause from pre-existing body cells, has nopurposeful function, and is characterized by a tendency to autonomousand unrestrained growth. Tumors are quite different from inflammatory orother swellings because the cells in tumors are abnormal in theirappearance and other characteristics. Abnormal cells, i.e. the kind ofcells that generally make up tumors, differ from normal cells in havingundergone one or more of the following alterations: (1) hypertrophy, oran increase in the size of individual cells; (2) hyperplasia or anincrease in the number of cells within a given zone; (3) anaplasia, or aregression of the physical characteristics of a cell toward a moreprimitive or undifferentiated type. Tumors may be benign, for examplelipomas, angiomas, osteomas, chondromas, and adenomas. Examples ofmalignant tumors are carcinomas (such as the breast tumors, carcinomasin the respiratory and gastrointestinal tracts, the endocrine glands,and the genitourinary system), sarcomas (in connective tissues,including fibrous tissues, adipose (fat) tissues, muscle, blood vessels,bone, and cartilage), carcinosarcoma (in both epithelial and connectivetissue) leukemias and lymphomas, tumors of nerve tissues (including thebrain), and melanoma (a cancer of the pigmented skin cells). The use ofthe binding proteins of the present invention against tumors can also bein combination with any other tumor therapy known in the art such asirradiation, photo-dynamic therapy, chemotherapy or surgery.

A pharmaceutical composition comprises binding proteins as describedabove and a pharmaceutically acceptable carrier, excipient or stabilizer(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]).Suitable carriers, excipients or stabilizers known to the skilled manare saline, Ringer's solution, dextrose solution, Hank's solution, fixedoils, ethyl oleate, 5% dextrose in saline, substances that enhanceisotonicity and chemical stability, buffers and preservatives. Othersuitable carriers include any carrier that does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids and amino acid copolymers. Apharmaceutical composition may also be a combination formulation,comprising an additional active agent, such as an anti-cancer agent oran anti-angiogenic agent (for example human VEGF-Axxxb; preferably,human VEGF-A165b).

A preferred pharmaceutical composition for the treatment of eye diseasescomprises binding proteins as described above and a detergent such asnonionic detergent, including but not limited to polysorbate 20 (e.g.about 0.04%), a buffer such as histidine, phosphate or lactic acid and asugar such as sucrose or trehalose. Preferably, such a compositioncomprises binding proteins as described above and PBS. Said or any otherpharmaceutical compositions described herein may be administeredlocally, either topically to a portion of the eye or be injected intothe eye for instance into the subconjunctivital, peri- or retrobulbarspace or directly into the eye. Alternatively, said or such otherpharmaceutical compositions may be administered systemically by parentaladministration. Preferably, said or such other pharmaceuticalcomposition is applied to the eye by an intravitreous injection. Alsopreferably, said pharmaceutical composition is applied to the eyetopically and as an eye drop. The eye drop may be applied to the cornea(clear part in the centre of the eye) thereby allowing the molecules topermeate into the eye. For the treatment of a disease affecting theposterior of the eye, it may be most desirable that the binding proteinpenetrates the sclera when injected under the conjunctiva or around theglobe. The administering of the binding protein may be performed after apreliminary step of modulating the surface of the eye to improvepenetration of the molecules. Preferably, the epithelial layer such asthe corneal epithelium is modulated by a penetration enhancer to allowfor a sufficient and rapid penetration of the molecules as for exampledescribed above. The use of the binding proteins of the presentinvention against eye diseases can also be in combination with any othertherapy known in the art such as photo-dynamic therapy.

The formulations to be used for in vivo administration must be asepticor sterile. This is readily accomplished by filtration through sterilefiltration membranes.

Sustained-release preparations may be prepared. In one embodiment of theinvention, an intraocular implant can be used for providing the bindingprotein of the invention. Suitable examples of sustained-releasepreparations include semipermeable matrices of solid hydrophobicpolymers containing a polypeptide of the invention, which matrices arein the form of shaped articles, e.g. films, or microcapsules. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate,non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolicacid copolymers such as the LUPRON DEPOT® (injectable microspherescomposed of lactic acid-glycolic acid copolymer and leuprolide acetate),and poly-D-(−)-3-hydroxybutyric acid.

The pharmaceutical composition may be administered by any suitablemethod within the knowledge of the skilled man. The preferred route ofadministration is parenterally. In parental administration, themedicament of this invention will be formulated in a unit dosageinjectable form such as a solution, suspension or emulsion, inassociation with the pharmaceutically acceptable excipients as definedabove. The dosage and mode of administration will depend on theindividual to be treated and the particular disease.

Generally, the pharmaceutical composition is administered so that thebinding protein of the present invention is given at a dose between 1μg/kg and 20 mg/kg, more preferably between 10 μg/kg and 5 mg/kg, mostpreferably between 0.1 and 2 mg/kg. Preferably, it is given as a bolusdose. Continuous infusion may also be used and includes continuoussubcutaneous delivery via an osmotic minipump. If so, the pharmaceuticalcomposition may be infused at a dose between 5 and 20 μg/kg/minute, morepreferably between 7 and 15 μg/kg/minute. In particular, thepharmaceutical composition is administered by injections into the eye sothat the binding protein of the invention is given at a dose between 0.1mg and 10 mg per injection, more preferably between 0.3 and 6 mg perinjection, most preferably between 1 mg and 4 mg per injection. Further,the pharmaceutical composition is administered by eye drops to the eyeso that a single drop of a solution containing a concentration of thebinding protein of the invention between 10 and 120 mg/ml, morepreferably between 20 and 100 mg/ml, most preferably between 40 and 80mg/ml is applied to the eye.

In another embodiment of the invention a binding protein inhibiting theactivity of VEGF-Axxx, as described above, can be used in combinationwith a binding protein or small molecule inhibiting the activity ofPlGF, with the same inhibition levels of PlGF as described above forVEGF-Axxx. This embodiment is based on the fact that PlGF is found to beangiogenic at sites where VEGF-Axxx levels are increased. Further, abinding protein inhibiting the activity of VEGF-Axxx, as describedabove, can be used in combination with a binding protein or smallmolecule inhibiting the activity of platelet-derived growth factor(PDGF), VEGF-C or other members of the VEGF family of proteins, tumornecrosis factor alpha (TNFalpha), delta-ligand like 4 (D114),interleukin 6 (IL-6), neuropilin or angiopoietin 2 (Ang2).

The invention further provides methods of treatment. In one aspect, amethod of treating a retinopathy is provided, the method comprisingadministering, to a patient in need thereof, a therapeutically effectiveamount of a binding protein of the invention, in particular a bindingprotein that inhibits the interaction between human VEGF-Axxx and humanVEGFR-2, but not the interaction between human VEGF-Axxxb and humanVEGFR-2, and the binding protein inhibits VEGFR-2 mediated angiogenesis.

The invention further relates to methods for using a binding protein asdescribed to inhibit a VEGF-A biological activity in a cell or toinhibit a biological activity mediated by VEGFR-2. The cell may besituated in vivo or ex vivo, and may be, for example, a cell of a livingorganism, a cultured cell or a cell in a tissue sample. The method maycomprise contacting said cell with any of the VEGF-A/VEGFR-2 interactioninhibiting binding proteins disclosed herein, in an amount and for atime sufficient to inhibit such biological activity.

The invention provides a method for treating a subject having acondition which responds to the inhibition of VEGF-Axxx or VEGFR-2. Sucha method comprises administering to said subject an effective amount ofa binding protein described herein. A condition may be one that ischaracterized by inappropriate angiogenesis. A condition may be ahyper-proliferative condition. Examples of conditions (or disorders)suitable for treatment include autoimmune disorders, inflammatorydisorders, retinopathies (particularly proliferative retinopathies), andcancers, in particular one of the diseases described above. Any of thebinding proteins described herein may be used for the preparation of amedicament for the treatment of such a disorder, particularly a disorderselected from the group consisting of: an autoimmune disorder, aninflammatory disorder, a retinopathy, and a cancer. Preferred conditions(or disorders) suitable for treatment are first-line metastatic renalcell carcinoma, relapsed glioblastoma multiforme, adjuvant colon cancer,adjuvant HER2-negative breast cancer, adjuvant HER2-positive breastcancer, adjuvant non-small cell lung cancer, diffuse large B-celllymphoma, first-line advanced gastric cancer, first-line HER2-negativemetastatic breast cancer, first-line HER2-positive metastatic breastcancer, first-line metastatic ovarian cancer, gastrointestinal stromaltumors, high risk carcinoid, hormone refractory prostate cancer, newlydiagnosed glioblastoma multiforme, metastatic head and neck cancer,relapsed platinum-sensitive ovarian cancer, second-line metastaticbreast cancer, extensive small cell lung cancer, non-squamous, non-smallcell lung cancer with previously treated CNS metastases and relapsedmultiple myeloma, prostate cancer, non-small cell lung cancer (NSCLC),colorectal cancer and pancreatic cancer, advanced ovarian cancer (AOC),AOC patients with symptomatic malignant ascites and non-Hodgkin'slymphoma.

The recombinant binding protein according to the invention may beobtained and/or further evolved by several methods such as display onthe surface of bacteriophages (WO 90/02809, WO 07/006665) or bacterialcells (WO 93/10214), ribosomal display (WO 98/48008), display onplasmids (WO 93/08278) or by using covalent RNA-repeat protein hybridconstructs (WO 00/32823), or intracellular expression andselection/screening such as by protein complementation assay (WO98/341120). Such methods are known to the person skilled in the art.

A library of ankyrin repeat proteins used for the selection/screening ofa recombinant binding protein according to the invention may be obtainedaccording to protocols known to the person skilled in the art (WO02/020565, Binz, H. K. et al., JMB, 332, 489-503, 2003, and Binz et al.,2004, loc. cit). The use of such a library for the selection VEGF-Axxxspecific DARPin® proteins is given in Example 1. In analogy, the ankyrinrepeat sequence motifs as presented above can used to build libraries ofankyrin repeat proteins that may be used for the selection or screeningof VEGF-Axxx specific DARPin® proteins. Furthermore, repeat domains ofthe present invention may be modularly assembled from repeat modulesaccording the current inventions and appropriate capping modules(Forrer, P., et al., FEBS letters 539, 2-6, 2003) using standardrecombinant DNA technologies (e.g. WO 02/020565, Binz et al., 2003, loc.cit. and Binz et al., 2004, loc. cit).

The invention is not restricted to the particular embodiments describedin the Examples. Other sources may be used and processed following thegeneral outline described below.

EXAMPLES

All of the starting materials and reagents disclosed below are known tothose skilled in the art, and are available commercially or can beprepared using well-known techniques.

Materials

Chemicals were purchased from Fluka (Switzerland). Oligonucleotides werefrom Microsynth (Switzerland). Unless stated otherwise, DNA polymerases,restriction enzymes and buffers were from New England Biolabs (USA) orFermentas (Lithuania). The cloning and protein production strain was E.coli XL1-blue (Stratagene, USA). VEGF variants were from R&D Systems(Minneapolis, USA) or were produced in Chinese Hamster Ovary Cells or inPichia pastoris and purified according to standard protocols (Rennet, E.S. et al., European J. Cancer 44, 1883-94, 2008; Pichia expressionsystem from Invitrogen). Biotinylated VEGF variants were obtainedchemically via coupling of the biotin moiety to primary amines of thepurified VEGF variants using standard biotinylation reagents and methods(Pierce, USA).

Molecular Biology

Unless stated otherwise, methods are performed according to describedprotocols (Sambrook J., Fritsch E. F. and Maniatis T., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory 1989, NewYork).

Designed Ankyrin Repeat Protein Libraries

The N2C and N3C designed ankyrin repeat protein libraries are described(WO 02/20565; Binz et al. 2003, loc. cit.; Binz et al. 2004, loc. cit.).The digit in N2C and N3C describes the number of randomized repeatmodules present between the N-terminal and C-terminal capping modules.The nomenclature used to define the positions inside the repeat unitsand modules is based on Binz et al. 2004, loc. cit. with themodification that borders of the repeat modules and repeat units areshifted by one amino acid position. For example, position 1 of a repeatmodule of Binz et al. 2004 (loc. cit.) corresponds to position 2 of arepeat module of the current disclosure and consequently position 33 ofa repeat module of Binz et al. 2004, loc. cit. corresponds to position 1of a following repeat module of the current disclosure.

All the DNA sequences were confirmed by sequencing, and the calculatedmolecular weight of all described proteins was confirmed by massspectrometry.

Example 1: Selection of Binding Proteins Comprising a Repeat Domain withBinding Specificity for VEGF-Axxx

Using ribosome display (Hanes, J. and Plückthun, A., PNAS 94, 4937-42,1997) many designed ankyrin repeat proteins (DARPin® proteins) withbinding specificity for VEGF-Axxx were selected from the N2C or N3CDARPin® libraries described by Binz et al. 2004 (loc. cit.). The bindingof the selected clones toward specific (VEGF-Axxx) and unspecific (MBP,E. coli maltose binding protein) targets was assessed by crude extractELISA indicating that VEGF-Axxx binding proteins were successfullyselected (FIG. 1). The repeat domains of SEQ ID NO:1 to 7 constituteamino acid sequences of selected binding proteins comprising a repeatdomain with binding specificity for VEGF-Axxx. Sequence analysis ofselected binders revealed specific ankyrin repeat sequence motifsinherent to certain selected families of binders.

Selection of VEGF-Axxx Specific Ankyrin Repeat Proteins by RibosomeDisplay

The selection of VEGF-Axxx specific ankyrin repeat proteins wasperformed by ribosome display (Hanes and Plückthun, loc. cit.) using dogVEGF-A164 or human VEGF-A165 as target proteins, the library of designedankyrin repeat proteins as described (WO 02/020565, Binz et al., 2003,loc. cit. and Binz et al., 2004, loc. cit) and established protocols(Zahnd, C., Amstutz, P. and Plückthun, A., Nat. Methods 4, 69-79, 2007).Ribosome-display selection rounds were performed on dog or human VEGFvariants (including biotinylated variants immobilized over NEUTRAVIDIN®or streptavidin) with both the N2C and N3C DARPin® libraries usingestablished protocols (Binz et al. 2004, loc. cit.). The number ofreverse transcription (RT)-PCR cycles after each selection round wasconstantly reduced from 40 to 30, adjusting to the yield due toenrichment of binders. Four initial selection rounds on dog VEGF yieldedpools of nanomolar-affinity DARPin® proteins, as revealed by ELISA andSPR measurements of single clones. To find DARPin® proteins with furtherimproved affinities, additional off-rate selections were performed onbiotinylated human or dog VEGF immobilized over NEUTRAVIDIN® orstreptavidin, taking pools after the second and third initialribosome-display selection rounds, followed by an on-rate selectionround on human VEGF.

Selected Clones Bind Specifically to VEGF-Axxx as Shown by Crude ExtractELISA

Individual selected DARPin® proteins specifically binding VEGF-Axxx wereidentified by an enzyme-linked immunosorbent assay (ELISA) using crudeEscherichia coli extracts of DARPin® protein expression cells usingstandard protocols. Selected clones were cloned into the pQE30 (Qiagen)expression vector, transformed into E. coli XL1-Blue (Stratagene) andthen grown overnight at 37° C. in a 96-deep-well plate (each clone in asingle well) containing 1 ml growth medium (2YT containing 1% glucoseand 100 μg/ml ampicillin). 1 ml of fresh 2YT containing 50 μg/mlampicillin was inoculated with 100 μl of the overnight culture in afresh 96-deep-well plate. After incubation for 2 h at 37° C., expressionwas induced with IPTG (1 mM final concentration) and continued for 3 h.Cells were harvested, resuspended in 100 μl B-PERII (Pierce) andincubated for 15 min at room temperature with shaking. Then, 900 μlPBS-TB (PBS supplemented with 0.2% BSA, 0.1% TWEEN® 20, pH 7.4) wereadded and cell debris were removed by centrifugation. 100 μl of eachlysed clone were applied to a well of a NEUTRAVIDIN® coated MAXISORP®plate containing either a VEGF-Axxx variant or the unrelated MBPimmobilized via their biotin moiety and incubated for 1 h at RT. Afterextensive washing with PBS-T (PBS supplemented with 0.1% TWEEN® 20, pH7.4) the plate was developed using standard ELISA procedures using themonoclonal anti-RGS(His)₄ antibody (34650, Qiagen) as primary antibodyand a polyclonal goat anti-mouse antibody conjugated with alkalinephosphatase (A3562, Sigma) as secondary reagent. Binding was thendetected by using disodium 4-nitrophenyl phosphate (4NPP, Fluka) as asubstrate for alkaline phosphatase. The color development was measuredat 405 nm. The results from an example crude extract ELISA used toidentify DARPin® proteins binding to VEGF-Axxx is shown in FIG. 1.Screening of several hundred clones by such a crude cell extract ELISArevealed more than hundred different DARPin® proteins with specificityfor VEGF-Axxx. These binding proteins were chosen for further analysis.Examples of amino acid sequences of selected ankyrin repeat domains thatspecifically bind to VEGF-Axxx are provided in SEQ ID NO:1 to 7.

Deducing Repeat Sequence Motives from Selected Repeat Domains withBinding Specificity for VEGF-Axxx

The amino acid sequences of selected repeat domains with bindingspecificity for VEGF-Axxx were further analyzed by sequence analyzingtools known to the practitioner in the art (WO 02/020565; Forrer et al.,2003, loc. cit.; Forrer, P., Binz, H. K., Stumpp, M. T. and Plückthun,A., ChemBioChem, 5(2), 183-189, 2004). Nevertheless, in contrast to WO02/020565 where naturally occurring repeat motifs were used to deducerepeat sequence motifs, here the repeat sequence motifs were deducedfrom the repeat units of selected repeat domains with bindingspecificity for VEGF-Axxx. Thereby families of selected repeat domainscomprising a common repeat sequence motif were determined.

High Level and Soluble Expression of DARPin® Proteins

For further analysis, the selected clones showing specific VEGF-Axxxbinding in the crude cell extract ELISA as described above wereexpressed in E. coli XL1-blue cells and purified using their His-tagusing standard protocols. 25 ml of stationary overnight cultures (LB, 1%glucose, 100 mg/I of ampicillin; 37° C.) were used to inoculate 1 lcultures (same medium). At A(600)=0.7, the cultures were induced with0.5 mM IPTG and incubated at 37° C. for 4 h. The cultures werecentrifuged and the resulting pellets were resuspended in 40 ml ofTBS500 (50 mM Tris-HCl, 500 mM NaCl, pH 8) and sonicated. The lysate wasrecentrifuged, and glycerol (10% (v/v) final concentration) andimidazole (20 mM final concentration) were added to the resultingsupernatant. Proteins were purified over a Ni-nitrilotriacetic acidcolumn (2.5 ml column volume) according to the manufacturer'sinstructions (QIAgen, Germany). Up to 200 mg of highly soluble DARPin®proteins with binding specificity to VEGF-Axxx could be purified fromone litre of E. coli culture with a purity >95% as estimated fromSDS-15% PAGE. Such purified DARPin® proteins are used for furthercharacterizations.

Example 2: Determination of IC₅₀ Values of Selected DARPin® Proteinswith Binding Specificity to VEGF-Axxx in a Spheroid Outgrowth Assay

Addition of VEGF-Axxx to HUVEC spheroids embedded in collagen matricesleads to spheroid sprouting. Addition of an inhibitor of VEGF-Axxx willblock sprout formation, which can be quantified statistically by thenumbers and lengths of sprouts. By adding different concentration ofinhibitor and a constant amount of VEGF, the IC₅₀ can be determined.

Inhibition of Spheroid Sprouting by VEGF-Axxx Specific DARPin® Proteins

Spheroid outgrowth assays were done according to standard protocols(Korff et al., loc. cit.). DARPin® proteins with specificity forVEGF-Axxx were selected and purified to >96% purity as described inExample 1. Human umbilical vein cells were grown to confluency inmonolayer culture. After trypsinization, the cell suspension was placedin a hanging drop to form spheroids, i.e. approximately 500 organizedaggregated HUVECs. Spheroids were embedded in a collagen matrix andstimulated with VEGF-A165 to initiate sprout outgrowth. Sproutinginhibitors were added additionally to observe their effects on sproutinginhibition. Sprout numbers per spheroid and sprout lengths werequantified using a graphical software.

The results from two example spheroid sprouting assays are shown in FIG.2A (DARPin #30 with binding specificity for VEGF-Axxx) and FIG. 2B(DARPin NC, a negative control DARPin® protein with no bindingspecificity for VEGF-Axxx; e.g. DARPin E3_5 (Binz et al., 2005, loc.cit.). The best performing DARPin® proteins in this assay showed IC₅₀values in the range of 10 to 50 pM, while AVASTIN® (bevacizumab),LUCENTIS® (ranibizumab) and MACUGEN® (pegaptanib) showed IC₅₀ values inparallel experiments in the range of 150 and 500 pM.

Example 3: Determination of the Target Specificity of DARPin #27 inComparison to AVASTIN® (Bevacizumab) by Surface Plasmon ResonanceAnalysis

Dog VEGF-A164 or Dog VEGF-A164b were immobilized in a flow cell and theinteraction of DARPin #27 (the repeat domain of SEQ ID NO:1,corresponding to amino acids 1 to 159) and AVASTIN® (bevacizumab) withthe immobilized targets were analyzed.

Surface Plasmon Resonance (SPR) Analysis

SPR was measured using a ProteOn instrument (BioRad). The running bufferwas 20 mM HEPES, pH 7.4, 150 mM NaCl and 0.005% TWEEN® 20. About 1200 RUof dog VEGF-A164 or dog VEGF-A164b were immobilized on a GLC chip(BioRad). The interactions were measured at a flow of 60 μl/min with 5min buffer flow, 100 seconds injection of AVASTIN® (bevacizumab) orDARPin #27 at a concentration of 250 nM and an off-rate measurement of afew minutes with buffer flow. The signal of an uncoated reference cellwas subtracted from the measurements.

The results are shown in FIG. 3A (AVASTIN® interaction with dogVEGF-A164), FIG. 3B (AVASTIN® interaction with dog VEGF-A164b), FIG. 3C(DARPin #27 interaction with dog VEGF-A164) and FIG. 3D (DARPin #27interaction with dog VEGF-A164b). Whereas AVASTIN® (bevacizumab) clearlyinteracts with both immobilized VEGF isoforms, the DARPin #27 shows onlyinteraction with VEGF-A164 and not VEGF-A164b.

Example 4: In Vivo Efficacy of DARPin #30 in Inhibiting VEGF-A165 in aVascular Leakage Rabbit Model

Pegylated DARPin #30 (the repeat domain of SEQ ID NO:4 corresponding tothe amino acids 1 to 126) or LUCENTIS® (ranibizumab) is applied byintravitreal injection into an eye of a rabbit to test their efficacy toinhibit vascular leakage induced by a subsequent intravitreous injectionof human VEGF-A165.

Vascular Leakage Inhibition Measurements in Rabbits

At day 1 either PBS, PEGylated DARPin #30 (125 μg) or the equimolaramount of LUCENTIS® (ranibizumab) (162 μg) is applied by an intravitrealinjection into one eye of each rabbit (treated eye). At day 4 or day 30the treated eye of each rabbit was challenged by intravitreal injectionof 500 ng of human VEGF-A165. Both eyes of all animals were evaluated 48hours after the VEGF-A165 injection by measuring the fluorescein contentin all eyes 1 h after intravenous injection of sodium fluorescein (50mg/kg animal body weight, 10% (w/v) in 0.9% (w/v) saline solution). Theratios of the amounts of fluorescence in the treated and untreated eyeswere calculated for every animal. A ratio of one corresponds to absenceof additional fluorescence leakage in the treated eye, a ratio greaterthan one indicates more fluorescence leakage in the treated eye than inthe untreated control eye.

Preparation of PEGylated DARPin® Protein

The PEGylation of protein by making use of a single Cys residue andmaleimide chemistry is well known to the person skilled in the art andcan be performed according to established protocols (e.g. from Pierce).DARPin #30 comprising an additional C-terminal linker (GGGSGGGSC, SEQ IDNO:8) was purified to near homogeneity using standard chromatographicmethods. The protein is completely reduced using DTT and purified bygel-filtration to remove the DTT and to exchange the buffer by PBS.PEG-maleimide (methoxy-poly(ethylene glycol)-oxopropylamino-propylmaleimide; NOF, no. SUNBRIGHT® ME-200MA) dissolved in PBS is mixed withthe DARPin® protein in PBS at about 15% molar excess of PEG-maleimidefor 2-4 hours at room temperature. The PEGylated DARPin® protein is thenseparated from non-reactive DARPin® protein and non-reactive PEGmoieties by using standard anion exchange chromatography.

The results are shown in FIG. 4. Both PEGylated DARPin #30 and LUCENTIS®(ranibizumab) were able to protect the rabbit eye from VEGF-A165 inducedvascular leakage 4 days after they were applied by intravitrealinjections. Nevertheless, only the PEGylated DARPin #30, and notLUCENTIS® (ranibizumab), was able to protect the rabbit eye fromVEGF-A165 induced vascular leakage up to 30 days after the intravitrealinjection.

In other experiments the intravitreal terminal half-lives of thedifferent binding proteins of the invention were measured afterintravitreal injections into rabbit eyes. DARPin #30 comprising anadditional C-terminal linker (GGGSGGGSC, SEQ ID NO:8) was conjugated toa 20 kDa and a 40 kDa non-proteinaceous PEG moiety using the respectivemaleimide PEGs from NOF (see Example 5). The terminal half-lives weredetermined to be 3.5 days (+/−0.3 days), 6.1 days (+/−1.0 days) and 5.4days (+/−0.8 days) for the DARPin #30, the DARPin #30 conjugated to the20 kDa PEG moiety and the DARPin #30 conjugated to the 40 kDA PEGmoiety. Surprisingly, increasing the molecular weight of thenon-proteinaceous PEG moiety from 20 kDa to 40 kDa did not result in anincreased terminal half-live. The same trend was observed incorresponding experiments were binding proteins comprising the repeatdomain of SEQ ID NO:1 (amino acids 1 to 159) or SEQ ID NO:3 (amino acids1 to 126) instead of the repeat domain of SEQ ID NO:4 were used.

Example 5: Recombinant Binding Proteins

Examples of recombinant binding proteins comprising a repeat domainbinding VEGF-Axxx and a proteinaceous polymer moiety are SEQ ID NO:1 and4. The repeat domain of SEQ ID NO:1 corresponds to amino acids 1 to 159and the proteinaceous polymer moiety of SEQ ID NO:1 corresponds to aminoacids 160 to 1′024. The repeat domain of SEQ ID NO:4 corresponds toamino acids 1 to 126 and the proteinaceous polymer moiety of SEQ ID NO:4corresponds to amino acids 127 to 536.

The binding proteins of SEQ ID NO:1 and 4 were expressed in thecytoplasm of Escherichia coli using standard techniques known to theperson skilled in the art (see, for example, the pQE expression systemfrom Qiagen (Germany)). The Met residue additionally encoded by theexpression vector was efficiently cleaved off in the cytoplasm of E.coli from the expressed polypeptide since the start Met is followed by asmall Gly residue (i.e. the amino acid at position 1 of SEQ ID NO:1 and4). The cells were lysed (e.g. by using a French press) and the bindingproteins were purified to near homogeneity from the crude cell extractby using standard chromatographic techniques known to the person skilledin the art.

Examples of recombinant binding proteins comprising one repeat domainbinding VEGF-Axxx and one non-proteinaceous polymer moiety were producedusing the repeat proteins of SEQ ID No: 2, 3, 5, 6, and 7. These repeatproteins comprise an N-terminal repeat domain, followed by a polypeptidelinker and a C-terminal Cys. The respective repeat domains correspond toamino acids 1 to 159 for SEQ ID NO:2 and 7, and to amino acids 1 to 126for SEQ ID NO:3 to 6. The repeat proteins of SEQ ID NO:2, 3, 5, 6, and 7were expressed in the cytoplasm of Escherichia coli using standardtechniques known to the person skilled in the art (see, for example, TheExpressionist from Qiagen (Germany)). The Met residue additionallyencoded by the expression vector was efficiently cleaved off in thecytoplasm of E. coli from the expressed polypeptide since the start Metis followed by a small Gly residue (i.e. the amino acid at position 1 ofSEQ ID NO:2, 3, 5, 6, and 7). The cells were lysed (e.g. by using aFrench press) and the binding proteins were purified to near homogeneityfrom the crude cell extract by using standard chromatographic techniquesknown to the person skilled in the art.

The purified repeat proteins comprising a single Cys residue were thenconjugated to a non-proteinaceous polymer moiety using standardmaleimide chemistry as outlined in Example 4. Thereby, a binding proteinof the invention comprising the repeat protein of SEQ ID NO:2 and a 40kDa non-proteinaceous PEG moiety (e.g. a 40 kDa maleimide-PEG(α-[3-(3-maleimido-1-oxopropyl)amino]propyl-ω-methoxy-polyoxyethylene)from NOF, product no. SUNBRIGHT® ME-400MA), the repeat protein of SEQ IDNO:3 and a 20 kDa non-proteinaceous PEG moiety (e.g. a 20 kDamaleimide-PEG(α-[3-(3-maleimido-1-oxopropyl)amino]propyl-ω-methoxy-polyoxyethylene)from NOF, product no. SUNBRIGHT® ME-200MA), the repeat protein of SEQ IDNO:5 and a 12 kDa non-proteinaceous PEG moiety (e.g. a 12 kDamaleimide-PEG(α-[3-(3-maleimido-1-oxopropyl)amino]propyl-ω-methoxy-polyoxyethylene)from NOF, product no. SUNBRIGHT® ME-120MA), the repeat protein of SEQ IDNO:6 and a 5 kDa non-proteinaceous PEG moiety (e.g. a 5 kDamaleimide-PEG(α-[3-(3-maleimido-1-oxopropyl)amino]propyl-ω-methoxy-polyoxyethylene)from NOF, product no. SUNBRIGHT® ME-050MA) and the repeat protein of SEQID NO:7 and a 2 kDa non-proteinaceous PEG moiety (e.g. a 2 kDamaleimide-PEG(α-[3-(3-maleimido-1-oxopropyl)amino]propyl-ω-methoxy-polyoxyethylene)from NOF, product no. SUNBRIGHT® ME-020MA) were produced. The PEGylatedrepeat proteins were then further separated from non-PEGylated repeatproteins and excess PEG by standard chromatographic techniques known tothe person skilled in the art.

Thus, SEQ ID NO:2, 3, 5, 6, and 7 were conjugated at the thiol of theirC-terminal cysteine to a maleimide PEG(α-[3-(3-maleimido-1-oxopropyl)amino]propyl-ω-methoxy-polyoxyethylene).The following structure was thereby produced:

wherein X is SEQ ID NO: 2, 3, 5, 6, or 7; and n is a positive integer.

1-15. (canceled)
 16. A method of treating a pathological condition in amammal including a human, comprising administering to a mammal in needthereof an effective amount of a recombinant binding protein, whereinthe pathological condition is a condition which responds to theinhibition of vascular endothelial growth factor-A165 (VEGF-A165) orvascular endothelial growth factor receptor-2 (VEGFR-2), and whereinsaid binding protein comprises an ankyrin repeat domain and apolyethylene glycol, wherein said ankyrin repeat domain is selected fromthe group consisting of (1) amino acids 1 to 159 of SEQ ID NO:1, (2)amino acids 1 to 159 of SEQ ID NO:2, (3) amino acids 1 to 126 of SEQ IDNO:3, (4) amino acids 1 to 126 of SEQ ID NO:4, (5) amino acids 1 to 126of SEQ ID NO:5, (6) amino acids 1 to 126 of SEQ ID NO:6, and (7) aminoacids 1 to 159 of SEQ ID NO:7.
 17. The method of claim 16, wherein saidpathological condition is pathological angiogenesis or vascular leakage.18. The method of claim 16, wherein said pathological condition is avascular disease.
 19. The method of claim 18, wherein said vasculardisease is a vascular disease of the eye.
 20. The method of claim 18,wherein said pathological condition is an ocular neovascularizationdisease.
 21. The method of claim 16, wherein said pathological conditionis a retinal ischemic disease.
 22. The method of claim 16, wherein saidpathological condition is cancer.